Immunoassay of PIVKA-II

ABSTRACT

The present invention provides an immunoassay for specifically measuring with high sensitivity PIVKA-II in serum or plasma through antigen-antibody reaction by adding an animal serum containing thrombin and/or an antibody reacting with human fibrin-like related substances to the reagents. The immunoassay of the invention comprises the steps of adding thrombin and/or an antibody reacting with human fibrin-like related substances to the reagents, and measuring PIVKA-II in serum or plasma.

This application is a 371 of PCT/JP00/03550 filed Jun. 1, 2000.

TECHNICAL FIELD

The present invention relates to an immunoassay utilizing anantigen-antibody reaction for specifically measuring with highsensitivity PIVKA-II (Protein Induced by Vitamin K Absence orAntagonist-II) in serum or plasma by adding thrombin and/or an antibodyreacting with human fibrin-like related substances to the reagents.

BACKGROUND ART

Along with AFP (α-fetoprotein), PIVKA-II (Protein Induced by Vitamin KAbsence or Antagonist-II) is measured widely in clinical examinationlaboratories as a hepatic cell tumor detecting marker which specificallyincreases in hepatic cell cancer patients. Generally, magnetic beads,glass beads, plastic plates, latexes or the like on which PIVKA-IIspecific monoclonal or polyclonal antibodies are adsorbed are subjectedto a first reaction with serum or plasma. Then, after washing thereaction mixture for B/F separation, a second reaction is carried out byadding human prothrombin specific polyclonal or monoclonal antibodieslabeled with an enzyme, a fluorescent material, a radioisotope, an Rucomplex or the like. Then, after washing the reaction mixture for B/Fseparation, the absorbance or luminescence of the enzyme, thefluorescent material, the radioisotope or the Ru bound to an immunecomplex formed by the antigen-antibody reaction is measured to determinePIVKA-II in the serum or the plasma.

Heretofore, PIVKA-Il has been measured by an enzyme immunoassay (EIA),but the EIA has poor sensitivity with a low positive rate for arelatively small hepatoma. Accordingly, an electrochemiluminescenceimmunoassay (ECLIA) utilizing an antigen or an antibody which is labeledwith an Ru complex was recently developed for a further highly sensitivemeasurement. The application of the electrochemiluminescence immunoassayled successfully to higher sensitivity in the PIVKA-II measurement. Torealize higher sensitivity not only in the ECLIA, but also in an enzymeimmunoassay, a chemiluminescence assay, a radioisotope assay, latexturbidimetry or the like, the influence of an unspecific reaction in asample should be taken into consideration.

In the process of studies for eliminating the influence of theunspecific reaction in a sample in the PIVKA-II measurement, it has beenfound that sensitivity and specificity of the measurement could beimproved by adding to the reagents, thrombin and/or an antibody thatreacts with a sensitivity with human fibrin-like related substances. Asthe substances attributable to such an unspecific reaction in thesample, attention was directed first to fibrin or its related substancesin the sample, and second to thrombin bound to fibrin or its relatedsubstances. In particular, when a polyclonal antibody is used as ananti-human prothrombin antibody for a second antibody or a labeledantibody, it may be subject to the interference of these unspecificreaction substances, thereby causing positive errors in measurement ofPIVKA-II. It is reported that the protein structure of prothrombin iscomposed of an F₁ fragment, an F₂ fragment and thrombin. The labeledantibody used for measurement of PIVKA-II may be not only ananti-prothrombin antibody, but also an anti-F₁ antibody, an anti-F₂antibody, or an anti-(F₁+F₂) antibody. However, in consideration of thepurity of these antibodies or the similarity of thrombin to the antigen,these antibodies may also react with bound or free thrombin in a sample.Further, in measurement of PIVKA-II, fibrin or its insoluble relatedsubstances in a sample are physically adsorbed onto carriers such asmagnetic beads, glass beads, latexes, plastic plates or the like to giverise to the phenomenon of positive errors in the measurement.

To prevent the interference attributable to fibrin-like relatedsubstances and thrombin, antibodies reacting with the human fibrin-likerelated substances, for example, anti-fibrinogen or anti-fibrinantibodies, and/or thrombin are added to the reagents. Thus, the presentinvention succeeds in accurately measuring a very small amount ofPIVKA-II by effectively inhibiting the nonspecific reaction.

An object of the present invention is to provide an immunoassayutilizing an antigen-antibody reaction for specifically measuring with ahigh sensitivity PIVKA-II in serum or plasma by adding to reagentsthrombin and/or an antibody reacting with human fibrin-like relatedsubstances to the reagents.

DISCLOSURE OF THE INVENTION

To solve the problems described above, an immunoassay for measuringPIVKA-II according to the present invention comprises the steps ofadding thrombin and/or an antibody reacting with human fibrin-likerelated substances to the reagents, and measuring PIVKA-II in serum orplasma.

The thrombin described above is preferably a thrombin-containing animalserum and/or purified thrombin, which may be heated or unheated.

In the present invention, the antibodies reacting with human fibrin-likerelated substances include, by way of example, anti-fibrinogenantibodies, anti-fibrin antibodies or the like, which are preferablypolyclonal antibodies, especially those highly reactive with not onlyfibrinogen or fibrin, but also fibrin-like related substances, such asFDP, fibrinopeptide A or fibrinopeptide B. As for the thrombin, purifiedpreparations are derived from human beings or animals, such as cows,pigs, sheep, horses, rabbits and chickens. Further, the use of a widevariety of thrombin-containing animal serums, such as bovine serum,sheep serum, porcine serum, horse serum, chicken serum and rabbit serumderived from animals different from the species of animals immunized forobtaining the labeled antibodies or second antibodies may lead toreaction inhibition of anti-thrombin antibodies occurring as impuritiesin the labeled antibodies.

As the labeled antibodies or second antibodies used in the presentinvention, it is possible to use not only human polyclonal antibodiesagainst prothrombin, F₁, F₂, or F₁+F₂, but also human monoclonalantibodies against prothrombin, F₁, F₂, or F₁+F₂. Here, F₁ and F₂ arepeptides constituting prothrombin. Polyclonal or monoclonal antibodiesprepared by immunization with synthetic peptides having the antigenicityof prothrombin can also be used.

The Examples of the present specification show application to anelectrochemiluminescence immunoassay. The present invention is alsouseful in a chemiluminescence assay, a radioisotope assay or the like toachieve a higher sensitivity. In the present invention, antibodies suchas anti-fibrinogen antibody and anti-fibrin antibody reacting withfibrin-like related substances such as fibrinogen, fibrin, FDP,fibrinopeptide A and fibrinopeptide B are preferably obtained byimmunization with human-derived fibrin-like related substances, butantibodies obtained by immunization with fibrin-like related substancessuch as animal-derived fibrinogen, fibrin or the like, andcross-reacting with human-derived fibrin-like related substances canalso be used. Antibodies, such as anti-fibrinogen or anti-fibrinantibodies specific to fibrin-like related substances, are preferablyadded to a reaction solution in the first reaction of the 2-stepsandwich method. On the other hand, thrombin is preferably added to thelabeled antibody solution or the second antibody solution in the secondreaction, the amount added thereof being preferably 1 to 50 NIH/ml.Those antibodies specific to fibrin-like related substances, such asanti-fibrinogen or anti-fibrin antibodies, a purified thrombin, and ananimal serum containing thrombin may be used singly or jointly as theoccasion demands.

The animal serums containing thrombin, such as bovine serum, sheepserum, porcine serum, horse serum, chicken serum and rabbit serum arederived from animals different from the species of animals immunized forobtaining the labeled antibodies or second antibodies, and may bepreferably added in an amount of 1 to 20%. These animal serums arederived from different species of animals and may be blended, whennecessary, with animal serums derived from the same species of animalsimmunized for obtaining the labeled antibody or second antibody.

If the enzyme activity of thrombin is strong when adding thrombin toreagents, the immune reaction may be adversely affected. If animalserums are added to those reagents containing the labeled antibodies orsecond antibodies, the stability of the reagents may be adverselyaffected. Therefore, a protease inhibitor for inhibiting the enzymeactivity of thrombin is preferably added to the reagents to whichthrombin or animal serums are added.

As the protease inhibitor, it is possible to use inhibitors, mentionedon page 452 in “Rinsho Koso Handbook (Clinical Enzyme Handbook)” (1sted., edited by Kitamura, Baba et al. and issued by Kodansha ScientificCo., on Sep. 10, 1982), such as, plasma proteinous inhibitors, hirudine,benzamidine and synthetic inhibitor such as PMSF (phenylmethylsulfonylfluoride), NPGB and or the like. However, these inhibitors are notsufficient for inhibiting the enzyme activity of thrombin, so it hasbeen found that the enzyme activity is significantly reduced withoutlosing its antigenicity even when a purified preparation of thrombin issubjected to heat treatment, e.g., at about 40 to 65° C.

A commercial purified preparation of thrombin is to be stored primarilyin a refrigerated or frozen form and not to be exposed to a hightemperature; The heating temperature for thrombin used in the presentinvention is 30 to 70° C., particularly preferably 40 to 60° C., so thatthe heating time can be reduced to 15 to 60 minutes. As a matter ofcourse, this heating is aimed at inactivating the enzyme activity ofthrombin, and hence insofar as the enzyme can be inactivated withoutlosing its antigenicity, the heating temperature and heating time arenot limited to the above ranges.

If the animal serum derived from animals of species different fromanimals immunized for the labeled antibody is previously heated for useas thrombin, the heating temperature is preferably 50 to 65° C. and theheating time is preferably 15 to 60 minutes. However, the heating timeand heating temperature, needless to say, can be regulated withoutparticular limitation in case of need. Further, the animal serum can beused without heating, if necessary.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention is described by reference to theExamples, but these Examples are shown for illustrative purposes onlyand are not construed as restrictive.

EXAMPLE 1 An Example of Measurement by an ElectrochemiluminescenceImmunoassay in an Automatic Analyzer Picolumi 8220

After 50 μl of a sample was added to 150 μl of a reaction solution, 25μl of magnetic beads having anti-PIVKA-II monoclonal antibodyimmobilized thereon were added thereto. After they were reacted at 30°C. for 9 minutes, 350 μl of a Picolumi BF washing solution (10 mM Trisbuffer) was added, and the magnetic beads trapped by a magnet werewashed 3 times. Then, 200 μl of Ru-labeled antibody solution containing1 μg/ml of a Ru-labeled anti-human prothrombin antibody (derived fromrabbit) was added to the magnetic beads, and these were allowed to reactat 30° C. for 9 minutes. Likewise, the magnetic beads trapped by amagnet were washed 3 times with the Picolumi BF washing solution. Afteraddition of 300 μl of a Picolumi luminescent electrolytic solutioncontaining 0.1 M tripropyl amine, the magnetic beads were sent to thesurface of an electrode and the luminescence of Ru bound to the magneticbeads was measured, the amount of PIVKA-II in the sample beingdetermined.

Reagent Composition

-   Reaction solution: 50 mM Tris buffer (pH 7.8), 0.150 M NaCl, 0.01%    Tween 20, 0.1% NaN₃, 5% rabbit serum (heated).-   Ru-labeled antibody solution: 50 mM Tris buffer (pH 7.8), 0.150 M    NaCl, 0.01% Tween 20, 0.1% NaN₃, 1 mM PMSF, 1 μg/ml Ru-labeled    anti-human prothrombin antibody (derived from rabbit), 5% rabbit    serum (heated).    (Preparation of Solid-Phase Magnetic Beads Having Anti-PIVKA-II    Monoclonal Antibody)

1 ml of 30 mg/ml magnetic beads (4.5 microns) was placed into a testtube and trapped with a magnet. After the supernatant was discarded, 1ml of 0.5 mg/ml anti-PIVKA-II monoclonal antibody (in 150 mM phosphatebuffer, pH 7.8) was added to the magnetic beads, and these were allowedto react at room temperature for 1 day under stirring. After themagnetic beads were washed, 2 ml of 1% BSA-Phosphate buffer was addedthereto, and the magnetic beads were blocked for 1 day under stirring atroom temperature. In case of use, the magnetic beads were diluted to 1mg/ml with the 1% BSA-Phosphate buffer.

(Preparation of Ru-Labeled Anti-Human Prothrombin Antibody)

68 μl of Ru-complex compound of ruthenium-tri-dipyridyl modified with asuccinimide group was added to 1 ml of 1 mg/ml anti-human prothrombinantibody immunized with rabbits, and these were allowed to react for 30minutes under stirring at room temperature. Then, the reaction wasterminated by adding 50 μl of 2 M glycine, and further the sample wasallowed to react for 10 minutes under stirring at room temperature.Finally, the sample was applied onto Sephadex G-25 (previouslyequilibrated with 10 mM phosphate buffer), and fractions of Ru-boundprotein were collected. The Ru-labeled anti-human prothrombin antibodythus obtained was diluted to 1 μg/ml in case of use.

180 μg/ml of anti-human fibrinogen antibody (derived from rabbit) wasadded to each reaction solution, a control solution without theanti-human fibrinogen antibody (derived from rabbit) and 8 human serumsbeing used to compare their specificity. Each serum was measured at n=3,and the results are shown in Table 1. Those reagents with the anti-humanfibrinogen antibody (derived from rabbit) show low dispersion inmeasured values and the absence of unspecific reaction.

TABLE 1 mAU/ml Addition of anti-fibrinogen Control C.V. antibody (180μg/ml) C.V. 1 56 38 98 50.0% 26 23 24 6.3% 2 47 45 35 15.2% 27 27 282.1% 3 37 35 30 10.6% 26 33 29 12.0%  4 55 21 25 55.2% 20 22 19 7.5% 527 37 44 23.7% 15 19 15 14.1%  6 21 23 26 10.8% 19 19 20 3.0% 7 22 31 2220.8% 21 20 18 7.8% 8 30 24 24 13.3% 24 24 24 0.0%

EXAMPLE 2

In this example, the same reagent composition in Example 1 was usedexcept that 10 NIH/ml of a purified preparation of bovine thrombin orhuman thrombin was added to the Ru-labeled antibody solution. Thissample showing particularly highly unspecific reactions was selected andmeasured simultaneously for PIVKA-II at n=10. The results of this sampleand a control sample in which neither the bovine thrombin nor the humanthrombin was added are shown in Table 2.

When the purified preparation of bovine or human thrombin was added tothe reagent, the specificity of the sample was improved as compared withthe control sample. This sample serum was centrifuged at 3000 rpm for 10minutes and an obtained supernatant thereof showed 80 mAU/ml.

EXAMPLE 3

In this example, the same reagent composition in Example 1 was usedexcept that 180 μg/ml anti-human fibrinogen antibody (derived fromrabbit) was added to the reaction solution and 10 NIH/ml bovine thrombinwas added to the Ru-labeled antibody solution. This sample, which showeda highly unspecific reaction, was measured simultaneously for PIVKA-11at n=10. The results of this sample-and a control sample to whichneither the anti-human fibrinogen antibody (derived from rabbit) nor thebovine thrombin was added are shown in Table 2 with the results inExample 2. Addition of both the anti-human fibrinogen antibody (derivedfrom rabbit) and bovine thrombin leads to an increase in specificity ofPIVKA-II much more in comparison with addition of bovine thrombin alone.

TABLE 2 mAU/ml Use of both bovine thrombin Addition Addition of andanti-human of human bovine fibrinogen Control thrombin thrombin antibody1 365 121 99 97 2 219 83 145 80 3 158 83 112 79 4 209 88 107 89 5 202 95119 72 6 150 154 104 79 7 247 84 110 80 8 133 110 103 82 9 166 121 94 8510  245 92 103 100 Mean 209.4 103.1 109.6 84.3

EXAMPLE 4

500 NIH of a purified preparation of bovine thrombin was added to 1 mlof 50 mM Tris buffer (0.15 M NaCl, pH 7.8) and heated at 50° C. for 30minutes in a thermostatic water bath. In this example, the same reagentcomposition in Example 1 was used except that 180 μg/ml anti-humanfibrinogen antibody (derived from rabbit) was added to the reactionsolution and the heat-treated purified preparation of bovine thrombinwas added at a concentration of 5 NIH/ml to the Ru-labeled antibodysolution. Using this sample, which showed a high unspecific reaction,PIVKA-II was measured. The results of this sample and a control sampleto which neither the anti-human fibrinogen antibody (derived fromrabbit) nor the heated bovine thrombin was added are shown in Table 3.As is evident from the results in Table 3, the inhibitory effect of thesample solutions in this example against the unspecific reaction wasexhibited similarly in Example 3. The enzyme activity of this heatedthrombin, as measured using Chromozyme TH (Boehringer), was reduced to ⅕compared with that of the unheated thrombin.

TABLE 3 mAU/ml Use of both heated bovine thrombin and anti-human Controlfibrinogen antibody 1 133 105 2 154 87 3 152 92 4 219 132 5 150 52 6 10087 7 137 98 8 125 90 9 162 89 10  127 86 Mean 145.9 91.8

EXAMPLES 5 And 6

In this example, the same reagent composition in Example 1 was usedexcept that 5% unheated rabbit serum (a control where the finalconcentration of the rabbit serum was 10%), 5% unheated horse serum(Example 5), or 5% unheated sheep serum (Example 6) was added toRu-labeled antibody solution, respectively. This sample, which showed ahighly unspecific reaction was measured for its inhibitory effectagainst the unspecific reaction, and the results are shown in Table 4.

TABLE 4 mAU/ml Rabbit serum + Rabbit serum + Control horse serum sheepserum 1 208 114 114 2 190 104 109 3 196 107 120 4 179 135 136 5 242 170132 Mean 203 126 122

As compared with the control, the unspecific reaction was inhibited inthe sample to which the horse serum or sheep serum was added. Thissample, which showed a highly unspecific reaction was centrifuged at3000 rpm for 10 minutes, and a supernatant was obtained which showed 74mAU/ml.

Capability of Exploitation in Industry:

As described above, according to the present invention, PIVKA-II inserum or plasma can be measured specifically with high sensitivity byadding thrombin and/or an antibody reacting with human fibrin-likerelated substances to the reagents.

1. An immunoassay method for measuring PIVKA-II in a serum or plasmatest sample, comprising the steps of: (a) contacting the serum or plasmatest sample with a first immunoassay reagent comprising anti-PIVKA-IIantibodies and antibodies which specifically bind to a human fibrin or ahuman fibrinogen; (b) contacting the resultant sample with a secondimmunoassay reagent comprising labeled antibodies which bind PIVKA-II,wherein the labeled antibodies are at least one antibody selected fromthe group consisting of anti-human prothrombin antibody, anti-F₁antibody to the F₁ prothrombin fragment, anti-F₂ antibody to the F₂prothrombin fragment and anti-(F₁+F₂) antibody to the F₁ and F₂prothrombin fragments; and (c) measuring the labeled antibodies, therebymeasuring PIVKA-II in the serum or plasma test sample.
 2. Theimmunoassay method according to claim 1, wherein the second immunoassayreagent further comprises thrombin.
 3. The immunoassay method accordingto claim 2, wherein the thrombin is a thrombin-containing animal serumand/or a purified thrombin.